Ebola virus (EboV) and Marburg virus (MarV) are filamentous enveloped non-segmented negative sense RNA viruses representing the two genera that comprise the family Filoviridae. These viruses are important human pathogens with case fatality rates ranging from 55% to 90% for EboV and up to 90% for MarV. These agents are classified as Category A Priority Pathogens by the NIAID/NIH and CDC, and there are presently no approved active or passive interventions for exposure resulting from natural outbreak, laboratory accident, or deliberate misuse. Public health concern is based on both the emerging infectious disease status of these viruses and their potential use as biologic weapons. An effective prophylactic vaccine would find application with medical personnel and close contacts during outbreaks in endemic areas of sub-Saharan Africa, with laboratory workers engaged in filovirus research, and with military and civilian personnel threatened by weaponized filoviruses. The ideal vaccine to meet both the outbreak and bio-weapon scenarios would rapidly confer protection against all species of EboV and MarV with a single administration. Among the vaccine technologies investigated to date, a tri-valent filovirus vaccine vectored by recombinant vesicular stomatitis virus (rVSV) has shown the greatest potential as a single administration vaccine with the capacity to rapidly provide broad protection against EboV and MarV. Profectus BioSciences has developed a replication competent attenuated rVSV vaccine delivery platform (rVSVN4CT1) that: 1) retains the immunogenicity of vaccines based on the non-attenuated vector, 2) has been manufactured under cGMPs at commercial scale, and 3) has been judged by the FDA to be safe for human testing. Thus, this application proposes: 1) to confirm that an attenuated tri-valent rVSVN4CT1 vectored filovirus vaccine will protect monkeys against challenge with EboV and MarV, 2) compliantly prepare seed stocks of the three viruses in the vaccine under conditions that will support future manufacture under cGMPs, 3) use the compliant seed stocks to prepare tri-valent vaccine that will be tested in a GLP neuro-toxicology study in non-human primates to confirm safety, 4) use the compliant tri-valent vaccine in development studies to identify a lyophilized formulation with the stability characteristics required for practical field use, and 5) test the lyophilized tri-valent vaccine to confirm that it protects NHPs from EboV and MarV when administered pre-exposure and post-exposure. PUBLIC HEALTH RELEVANCE: Ebola virus (EboV) and Marburg virus (MarV) are filamentous enveloped non-segmented negative sense RNA viruses representing the two genera that comprise the family Filoviridae. These viruses are important human pathogens with case fatality rates ranging from 55% to 90% for EboV and up to 90% for MarV. These agents are classified as Category A Priority Pathogens by the NIAID/NIH and CDC, and there are presently no approved active or passive interventions for exposure resulting from natural outbreak, laboratory accident, or deliberate misuse. Public health concern is based on both the emerging infectious disease status of these viruses and their potential use as biologic weapons. An effective prophylactic vaccine would find application with medical personnel and close contacts during outbreaks in endemic areas of sub-Saharan Africa, with laboratory workers engaged in filovirus research, and with military and civilian personnel threatened by weaponized filoviruses. The studies under this application propose to develop and test a vaccine vectored with vesicular stomatitis virus that will protect against infection with EboV and MarV.